Natural IgM are highly represented in the circulation at birth, and these often autoreactive antibodies have been postulated to have innate-like properties and play crucial roles in apoptotic cell ...clearance, tissue homeostasis, and immune modulation. This review summarizes the known properties of these IgM autoantibodies, and the evidence that these anti-apoptotic cell IgM natural antibodies can regulate inflammatory responses through ancient pathways of the innate immune system that first arose long before the initial emergence of the adaptive immune system. While the regulatory contributions of these natural IgM autoantibodies are certainly not an essential and fundamental component of host defenses, these provide an additional layer to further protect the host. More importantly, these IgM antibody responses are highly inducible and their up-regulation can be a powerful means for the host to survive in a setting of chronic inflammation. The observed beneficial clinical associations for cardiovascular disease and autoimmunity, as well as opportunities for potential therapeutic implications are discussed.
Objective
Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely ...understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross‐reactivity.
Methods
IgG‐secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full‐length mAb (n = 93) and also as germline‐reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated.
Results
Four unrelated anti–citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell–derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay.
Conclusion
These findings suggest that the high level of cross‐reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.
Objective
Anti–citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease‐relevant autoantigens that are targeted by ACPAs remains a ...matter of debate. This study utilized patient‐derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA.
Methods
Twelve human monoclonal ACPAs (positive by the second‐generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head‐to‐head comparison of their reactivities was performed. For profiling, we used a complementary DNA–based protein array (Engine GmbH) and 3 peptide‐screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histone‐derived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products).
Results
The fine‐specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone‐distinct consensus binding motifs (e.g., Cit–Gly, Gly–Cit, or Arg–Cit–Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA‐binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for citrullinated peptides.
Conclusion
ACPAs and anti–modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti–carbamylated and anti–citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.
Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect ...additional mechanisms by which ACPAs might contribute to development of joint pathology.
Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting.
Challenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis.
We propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration.
The use of combinatorial protein engineering to design proteins with novel binding specificities and desired properties has evolved into a powerful technology, resulting in the recent advances in ...protein library selection strategies and the emerge of a variety of new engineered affinity proteins. The need for different protein library selection methods is due to that each target protein pose different challenges in terms of its availability and inherent properties. At present, alternative engineered affinity proteins are starting to complement and even challenge the classical immunoglobulins in different applications in biotechnology and potentially also for in vivo use as imaging agents or as biotherapeutics. This review article covers the generation and use of affinity proteins generated through combinatorial protein engineering. The most commonly used selection techniques for isolation of desired variants from large protein libraries are described. Different antibody derivatives, as well as a variety of the most validated engineered protein scaffolds, are discussed. In addition, we provide an overview of some of the major present and future applications for these engineered affinity proteins in biotechnology and medicine.
Proteins subjected to post-translational modifications, such as citrullination, carbamylation, acetylation or malondialdehyde (MDA)-modification are targeted by autoantibodies in seropositive ...rheumatoid arthritis (RA). Epidemiological and experimental studies have both suggested the pathogenicity of such humoral autoimmunity, however, molecular mechanisms triggered by anti-modified protein antibodies have remained to be identified.
Here we describe in detail the pathways induced by anti-MDA modified protein antibodies that were obtained from synovial B cells of RA patients and that possessed robust osteoclast stimulatory potential and induced bone erosion in vivo. Anti-MDA antibodies boosted glycolysis in developing osteoclasts via an FcγRI, HIF-1α and MYC-dependent mechanism and subsequently increased oxidative phosphorylation. Osteoclast development required robust phosphoglyceride and triacylglyceride biosynthesis, which was also enhanced by anti-MDA by modulating citrate production and expression of the glycerol-3-phosphate dehydrogenase 1 (GPD1) and glycerol-3-phosphate acyltransferase 2 (GPAT2) genes. In summary, we described novel metabolic pathways instrumental for osteoclast differentiation, which were targeted by anti-MDA antibodies, accelerating bone erosion, a central component of RA pathogenesis.
•Anti-MDA antibodies are inducers of osteoclast maturation and bone erosion in RA.•Anti-MDA antibodies boost glycolysis via FcγRI, HIF-1α and MYC in pre-osteoclast.•Osteoclast glycolysis fuels mitochondrial oxidation and overproduction of citrate.•Anti-MDA boosts lipid biosynthesis via regulating citrate and the GPD1/GPAT2 genes.•Glycerolipid biosynthesis is crucial for osteoclast development.
Axl is a receptor tyrosine kinase with important functions in immune regulation. We investigated serum levels of soluble (s)Axl in lupus nephritis (LN) in association with renal disease activity, ...tissue damage and treatment response. We surveyed 52 patients with International Society of Nephrology/Renal Pathology Society (ISN/RPS) class III/IV LN and 20 healthy controls. Renal biopsies were performed at the time of active LN and post-treatment. Patients were classified as clinical responders (CRs) or clinical non-responders based on the American College of Rheumatology (ACR) criteria. Improvement by ≥50% in renal activity index scores defined histological responders (HRs). sAxl levels were elevated in patients compared to controls (median: 18.9 ng/mL), both at baseline (median: 45.7; P<0.001) and post-treatment (median: 41.2 ng/mL; P<0.001). Baseline sAxl levels were higher in patients with class IV (median: 47.7 ng/mL) versus class III (median: 37.5 ng/mL) nephritis (P = 0.008), and showed moderate correlations with albuminuria (r = 0.30, P = 0.030) and creatinine (r = 0.35, P = 0.010). Baseline sAxl levels decreased in CRs (P = 0.002) and HRs (P<0.001), but not in non-responders; levels ≥36.6 ng/mL yielded a >5 times higher probability of histology-based response (odds ratio, OR: 5.5; 95% confidence interval, CI: 1.2-25.1). High post-treatment sAxl levels were associated with worsening in chronicity index scores (P = 0.025); low levels predicted favourable renal outcome (creatinine ≤88.4 μmol/L) 10 years after the baseline renal biopsy (area under the curve: 0.71; 95% CI: 0.54-0.89). In conclusion, sAxl may prove useful as a marker of renal activity, histological response to immunosuppression, and renal damage progression in LN. Persistently high sAxl levels after completion of treatment may be indicative of a need for treatment intensification.
Abstract Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are ...part of the natural antibody pool, while elevated levels of IgG anti-MDA antibodies are associated with inflammatory and autoimmune conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells, and 3.5% of memory B cells and 2.3% of plasma cells were found to be anti-MDA positive. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications such as citrullination, carbamylation or 4-HNE-carbonylation. The mAbs recognized MDA-adducts in a variety of proteins including albumin, histone 2B, fibrinogen and vimentin. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by near germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. Importantly, these anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses.
Objective
In rheumatoid arthritis (RA), autoreactive B cells are pathogenic drivers and sources of anti–citrullinated protein antibodies (ACPAs) that are a diagnostic biomarker and predictor of worse ...long‐term prognosis. Yet, the immunobiologic significance of persistent ACPA production at the cellular level is poorly understood. This study was undertaken to investigate the representation of ACPA‐expressing switched‐memory B cells in RA.
Methods
In a cross‐sectional study of RA patients, we investigated the presence of continued defects in immune homeostasis as a function of disease activity. Using an enzyme‐linked immunosorbent assay (ELISA) and a sensitive multiplex bead‐based immunoassay, we characterized fine binding antibody specificities in sera, synovial fluid (SF), and B cell culture supernatants. In this manner, we determined the frequency and epitope reactivity patterns of ACPAs produced by SF B cells and switched‐memory blood B cells and compared the latter to serum ACPA levels and disease activity scores.
Results
Cultured B cells from SF were shown to spontaneously secrete ACPAs, while constitutive IgG autoantibody production by peripheral blood mononuclear cells (PBMCs) was substantially less frequent. After in vitro stimulation, PBMCs secreted IgG ACPA that was overwhelmingly from switched‐memory B cells, across all patient groups treated with methotrexate and/or a tumor necrosis factor inhibitor. Intriguingly, the frequencies of ACPA‐expressing switched‐memory B cells significantly correlated with serum IgG anti–cyclic citrullinated peptide 3 (r = 0.57, P = 0.003). Moreover, treatment‐induced clinical remission had little or no effect on the circulating burden of switched‐memory ACPA‐expressing B cells, in part explaining the continued dysregulation of humoral immunity.
Conclusion
Our findings rationalize why therapeutic cessation most often results in disease reactivation and clinical flare. Hence, a clinical disease activity score is not a reliable indicator of the resolution of pathologic recirculating B cell autoimmunity.
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